The long-term objective of this proposal is to gain a better understanding of the molecular mechanisms of regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) subunit gene expression by pulsatile gonadotropin-releasing hormone (GnRH). The precise episodic timing of the release of the gonadotropins, LH and FSH, is pivotal for the control of sexual development and reproductive function. LH and FSH are synthesize and secreted from pituitary gonadotropes under regulation by the hypothalamic peptide, GnRH. GnRH is released. is released in a pulsatile fashion with the frequency of release varying developmentally and throughout the menstrual or estrous cycle. LH and FSH biosynthesis and secretion are differentially regulated by these changes in GnRH pulse frequency. The signal transduction pathways that are activated by these changes in GnRH frequency to specifically regulated the expression of the gonadotropin subunit genes remain poorly understood. The present research proposal focuses on the elucidation of the signal transduction pathways involved in mediating the differential effects of varying GnRH pulse frequencies on LH and FSH subunit gene expression. Using the recently developed, gonadotrope-derived LbetaT2 cell line exposed to GnRH at varying GnRH pulse frequencies, we plan to study the roles of G-protein alpha subunits, G-protein, beta/gamma subunits, and signal transduction pathways downstream of an activated by these G-proteins, in this differential regulation. Ultimately, we will perform confirmatory studies in primary rat pituitary cell cultures to validate the physiologic relevance of our findings. In addition, several naturally occurring mutations in the GnRH have been identified recently in patients with reduced fertility and idiopathic hypogonadotropic hypogonadism (IHH). We propose to examine the effects of these newly identified mutations on GnRH signaling and to correlate them with effects on GnRH-stimulated gonadotropin subunit gene expression. The identification and functional characterization of these mutations will provide a clinically relevant and important context in which to study pathways of GnRH action. Taken together, these studies will provide new insights into the mechanisms of regulation of gonadotropins by GnRH, which will, in turn, lead to a better understanding of reproductive development, and may guide the development of new and innovative therapies for disorders of reproductive function.